H3K14ac polyclonal antibody

Lot	A2283P
Concentration	0.9 µg/µl
Species reactivity	Human
Type	Polyclonal
Purity	Affinity purified
Host	Rabbit
Precautions	This product is for research use only. Not for use in diagnostic or therapeutic procedures.

Applications	Suggested dilution ^*	References
ChIP ^*	2 μg/ChIP	Fig 1
ELISA	1:1,000 - 1:5,000	Fig 3
Dot Blotting	1:10,000	Fig 3
Western Blotting	1:1,000	Fig 4

H3K14ac Antibody ChIP Grade

Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K14ac
ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K14ac (cat. No. C15410310) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. C01010055), using sheared chromatin from 1.5 million cells. A titration of the antibody consisting of 0.5, 1, 2 and 5 μg per ChIP experiment was analysed. IgG (1 μg/IP) was used as negative IP control. QPCR was performed with primers for a region approximately 1 kb upstream of the ACTB and GAPDH promoters, used as positive controls, and for the coding region of the inactive MYOD1 gene and the Sat2 satellite repeat, used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

H3K14ac Antibody ELISA validation

Figure 2. Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K14ac (cat. No. C15410310) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:73,200.

H3K14ac Antibody validated in Dot Blot

Figure 3. Cross reactivity tests using the Diagenode antibody directed against H3K14ac
To test the cross reactivity of the Diagenode antibody against H3K14ac (cat. No. C15410310), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H3K14. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:10,000. Figure 3 shows a high specificity of the antibody for the modification of interest.

H3K14ac Antibody validated in Western Blot

Figure 4. Western blot analysis using the Diagenode antibody directed against H3K14ac
Western blot was performed on whole cell (25 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H3K14ac (cat. No. C15410310). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

Target Description

Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Acetylation of histone H3 is associated with active genes.

実験手法

ELISA
Enzyme-linked immunosorbent assay. Read more

DB
Dot blotting Read more

WB
Western blot : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies. Learn more about: Load... Read more

ChIP-qPCR (ab)
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資料

Datasheet H3K14ac C15410310 DATASHEET
Datasheet description

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出版物